Mechanosensitivity is required for effective gastrointestinal function. The overall objective of this proposal is to understand the ionic mechanisms that underlie smooth muscle and interstitial cell of Cajal (ICC) mechanosensitivity, to determine the role of ion channelopathies in motility disorders and to establish new roles for ion channel interacting proteins and the lipid environment in the regulation of mechanosensory ion channels in health and in disease. The central hypothesis of this proposal is that mechanosensory ion channels expressed in human intestinal smooth muscle cells and ICC are regulated by specific mechanisms. The Na+ channel is gated by Na+ channel interacting proteins (SCIPs) and the L-type Ca2+ channel by the lipid bilayer. Mutations in the channel subunits themselves and/or the channel interacting proteins can contribute to motility disorders. The central hypothesis will be tested in two specific aims. Specific Aim 1 will test the hypothesis that the mechanosensitive Na+ current in human intestinal smooth muscle cells and ICC is regulated by SCIPs that make up a functional unit and that mutations in proteins that make up the functional unit can result in disease. Specific Aim 2 will test the hypothesis that mechanosensitivity of L-type Ca2+ channels is a result of an interaction between the channel and the lipid bilayer. The central hypothesis is supported by preliminary data that show that SCIPs, including telethonin, act in concert with the mechanosensory Na+ channel to create a functional unit, that mutations in the gene (SCN5A) that encodes for Nav1.5 result in gastrointestinal symptoms, that mutations in SCIPs, newly identified in this proposal, are found in patients with motility disorders, and that tension in the lipid bilayer mechanically gates the L-type Ca2+ channel and alters open probability. The PI will test the central hypothesis by the combination of electrophysiological, molecular, and population based techniques. These include patch clamp, immunohistochemistry, Western blots, RT-PCR, SCPCR, qRT-PCR, yeast two hybrid, GST-pulldowns, denaturing high performance liquid chromatography and questionnaire based techniques. Successful completion of the proposed studies has both basic significance and clinical impact. We are now poised to significantly advance our understanding of the components of a functional mechanosensitive Nav1.5 channel and on the mechanism of L-type Ca2+ channel mechanosensitivity. The work will also provide mechanistic information that is relevant to not only the gastrointestinal tract but also to organs such as the heart that also express mechanosensory ion channels and/or ion channel interacting proteins. Understanding the role mutations in ion channels and in ion channel interacting proteins play in motility disorders will not only help determine the cause of these disorders but will also serve to identify future therapeutic targets and strategies to treat motility disorders of the gastrointestinal tract.